2′-O-aminoethyloxyethyl-modified oligonucleotides

ABSTRACT

2′-O-Modified ribosyl nucleosides and modified oligomeric compounds containing such nucleosidic monomers are disclosed. Oligomeric compounds are disclosed that have increased binding affinity as shown by molecular modeling experiments. The 2′-O-modified nucleosides of the invention include ring structures that position the sugar moiety of the nucleosides preferentially in 3′ endo geometries.

RELATED APPLICATION DATA

This patent application is a continuation-in-part of application Ser.No. 09/130,566, filed Aug. 7, 1998, now U.S. Pat. No. 6,043,352 thecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to nucleosidic monomers and oligomericcompounds incorporating such nucleosidic monomers, and methods of usingsuch oligomeric compounds. The oligomeric compounds of the invention areuseful for therapeutic and investigative purposes. More specifically,the present invention is directed to the use of oligomeric compoundshaving 2′-O-modifications that will increase their affinity and nucleaseresistance.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals, includingmost disease states, are affected by proteins. Classical therapeuticmodes have generally focused on interactions with such proteins in aneffort to moderate their disease-causing or disease-potentiatingfunctions. Recently, however, attempts have been made to moderate theactual production of such proteins by interactions with molecules thatdirect their synthesis, such as intracellular RNA. By interfering withthe production of proteins, maximum therapeutic effect and minimal sideeffects may be realized. It is the general object of such therapeuticapproaches to interfere with or otherwise modulate gene expressionleading to undesired protein formation.

One method for inhibiting specific gene expression is the use ofoligonucleotides. Oligonucleotides are now accepted as therapeuticagents with great promise, and are known to hybridize to single-strandedDNA or RNA molecules. Hybridization is the sequence-specific base pairhydrogen bonding of nucleobases of the oligonucleotide to thenucleobases of the target DNA or RNA molecule. Such nucleobase pairs aresaid to be complementary to one another. The concept of inhibiting geneexpression through the use of sequence-specific binding ofoligonucleotides to target RNA sequences, also known as antisenseinhibition, has been demonstrated in a variety of systems, includingliving cells (see, e.g., Wagner et al., Science (1993) 260:1510-1513;Milligan et al., J. Med. Chem., (1993) 36:1923-37; Uhlmann et al., Chem.Reviews, (1990) 90:543-584; Stein et al., Cancer Res., (1988)48:2659-2668).

The events that provide the disruption of the nucleic acid function byantisense oligonucleotides (Cohen in Oligonucleotides: AntisenseInhibitors of Gene Expression, (1989) CRC Press, Inc., Boca Raton, Fla.)are thought to be of two types. The first, hybridization arrest, denotesthe terminating event in which the oligonucleotide inhibitor binds tothe target nucleic acid and thus prevents, by simple steric hindrance,the binding of essential proteins, most often ribosomes, to the nucleicacid. Methyl phosphonate oligonucleotides: Miller, P. S. and Ts'O,P.O.P. (1987) Anti-Cancer Drug Design, 2:117-128, and α-anomeroligonucleotides are the two most extensively studied antisense agentswhich are thought to disrupt nucleic acid function by hybridizationarrest.

The second type of terminating event for antisense oligonucleotidesinvolves the enzymatic cleavage of the targeted RNA by intracellularRNase H. A 2′-deoxyribofuranosyl oligonucleotide or oligonucleotideanalog hybridizes with the targeted RNA and this duplex activates theRNase H enzyme to cleave the RNA strand, thus destroying the normalfunction of the RNA. Phosphorothioate oligonucleotides are the mostprominent example of an antisense agent that operates by this type ofantisense terminating event.

Oligonucleotides may also bind to duplex nucleic acids to form triplexcomplexes in a sequence specific manner via Hoogsteen base pairing (Bealet al., Science, (1991) 251:1360-1363; Young et al., Proc. Natl. Acad.Sci. (1991) 88:10023-10026). Both antisense and triple helix therapeuticstrategies are directed towards nucleic acid sequences that are involvedin or responsible for establishing or maintaining disease conditions.Such target nucleic acid sequences may be found in the genomes ofpathogenic organisms including bacteria, yeasts, fungi, protozoa,parasites, viruses, or may be endogenous in nature. By hybridizing toand modifying the expression of a gene important for the establishment,maintenance or elimination of a disease condition, the correspondingcondition may be cured, prevented or ameliorated.

In determining the extent of hybridization of an oligonucleotide to acomplementary nucleic acid, the relative ability of an oligonucleotideto bind to the complementary nucleic acid may be compared by determiningthe melting temperature of a particular hybridization complex. Themelting temperature (T_(m)), a characteristic physical property ofdouble helices, denotes the temperature (in degrees centigrade) at which50% helical (hybridized) versus coil (unhybridized) forms are present.T_(m) is measured by using the UV spectrum to determine the formationand breakdown (melting) of the hybridization complex. Base stacking,which occurs during hybridization, is accompanied by a reduction in UVabsorption (hypochromicity). Consequently, a reduction in UV absorptionindicates a higher T_(m). The higher the T_(m), the greater the strengthof the bonds between the strands.

Oligonucleotides may also be of therapeutic value when they bind tonon-nucleic acid biomolecules such as intracellular or extracellularpolypeptides, proteins, or enzymes. Such oligonucleotides are oftenreferred to as “aptamers” and they typically bind to and interfere withthe function of protein targets (Griffin, et al., Blood, (1993),81:3271-3276; Bock, et al., Nature, (1992) 355: 564-566).

Oligonucleotides and their analogs (oligomeric compounds) have beendeveloped and used for diagnostic purposes, therapeutic applications andas research reagents. For use as therapeutics, oligonucleotidespreferably are transported across cell membranes or be taken up bycells, and appropriately hybridize to target DNA or RNA. These functionsare believed to depend on the initial stability of the oligonucleotidestoward nuclease degradation. A deficiency of unmodified oligonucleotideswhich affects their hybridization potential with target DNA or RNA fortherapeutic purposes is their degradation by a variety of ubiquitousintracellular and extracellular nucleolytic enzymes referred to asnucleases. For oligonucleotides to be useful as therapeutics ordiagnostics, the oligonucleotides should demonstrate enhanced bindingaffinity to complementary target nucleic acids, and preferably bereasonably stable to nucleases and resist degradation. For anon-cellular use such as a research reagent, oligonucleotides need notnecessarily possess nuclease stability.

A number of chemical modifications have been introduced intooligonucleotides to increase their binding affinity to target DNA or RNAand resist nuclease degradation. Modifications have been made, forexample, to the phosphate backbone to increase the resistance tonucleases. These modifications include use of linkages such as methylphosphonates, phosphorothioates and phosphorodithioates, and the use ofmodified sugar moieties such as 2′-O-alkyl ribose. Other oligonucleotidemodifications include those made to modulate uptake and cellulardistribution. A number of modifications that dramatically alter thenature of the internucleotide linkage have also been reported in theliterature. These include non-phosphorus linkages, peptide nucleic acids(PNA's) and 2′-5′ linkages. Another modification to oligonucleotides,usually for diagnostic and research applications, is labeling withnon-isotopic labels, e.g., fluorescein, biotin, digoxigenin, alkalinephosphatase, or other reporter molecules.

Over the last ten years, a variety of synthetic modifications have beenproposed to increase nuclease resistance, or to enhance the affinity ofthe antisense strand for its target mRNA (Crooke et al., Med. Res. Rev.,1996, 16, 319-344; De Mesmaeker et al., Acc. Chem. Res., 1995, 28,366-374). A variety of modified phosphorus-containing linkages have beenstudied as replacements for the natural, readily cleaved phosphodiesterlinkage in oligonucleotides. In general, most of them, such as thephosphorothioate, phosphoramidates, phosphonates and phosphorodithioatesall result in oligonucleotides with reduced binding to complementarytargets and decreased hybrid stability.

RNA exists in what has been termed “A Form” geometry while DNA exists in“B Form” geometry. In general, RNA:RNA duplexes are more stable, or havehigher melting temperatures (Tm) than DNA:DNA duplexes (Sanger et al.,Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York,N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al.,Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNAhas been attributed to several structural features, most notably theimproved base stacking interactions that result from an A-form geometry(Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presenceof the 2′ hydroxyl in RNA biases the sugar toward a C3′ endo pucker,i.e., also designated as Northern pucker, which causes the duplex tofavor the A-form geometry. On the other hand, deoxy nucleic acids prefera C2′ endo sugar pucker, i.e., also known as Southern pucker, which isthought to impart a less stable B-form geometry (Sanger, W. (1984)Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.).In addition, the 2′ hydroxyl groups of RNA can form a network of watermediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,Biochemistry, 1996, 35, 8489-8494).

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes and, depending on their sequence, may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996,264, 521-533). The stability of a DNA:RNA hybrid a significant aspect ofantisense therapies, as the proposed mechanism requires the binding of amodified DNA strand to a mRNA strand. Ideally, the antisense DNA shouldhave a very high binding affinity with the mRNA. Otherwise, the desiredinteraction between the DNA and target mRNA strand will occurinfrequently, thereby decreasing the efficacy of the antisense.oligonucleotide.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the 2′-methoxyethoxy(MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997,272, 11944-12000; Freier et al., Nucleic Acids Res., 1997, 25,4429-4443). One of the immediate advantages of the MOE substitution isthe improvement in binding affinity, which is greater than many similar2′ modifications such as O-methyl, O-propyl, and O-aminopropyl (Freierand Altmann, Nucleic Acids Research, (1997) 25:4429-4443).2′-O-Methoxyethyl-substituted also have been shown to be antisenseinhibitors of gene expression with promising features for in vivo use(Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al.,Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996,24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16,917-926). Relative to DNA, they display improved RNA affinity and highernuclease resistance. Chimeric oligonucleotides with2′-O-methoxyethyl-ribonucleoside wings and a centralDNA-phosphorothioate window also have been shown to effectively reducethe growth of tumors in animal models at low doses. MOE substitutedoligonucleotides have shown outstanding promise as antisense agents inseveral disease states. One such MOE substituted oligonucleotide ispresently being investigated in clinical trials for the treatment of CMVretinitis.

Although the known modifications to oligonucleotides, including the useof the 2′-O-methoxyethyl modification, have contributed to thedevelopment of oligonucleotides for various uses, there still exists aneed in the art for further modifications that will impart enhancedhybrid binding affinity and/or increased nuclease resistance tooligonucleotides and their analogs.

SUMMARY OF THE INVENTION

The present invention provides oligomeric compounds having at least one2′—O—CH₂CH₂—O—CH₂CH₂—N (R₁)(R₂) modified nucleoside. Preferredoligomeric compounds of the invention are those that include at leastone nucleoside of the formula:

wherein

Bx is a heterocyclic base;

each R₁ and R₂ is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, whereinsaid substitution is OR₃, SR₃, NH₃ ⁺, N(R₃)(R₄), guanidino or acyl wheresaid acyl is an acid, amide or an ester;

or R₁ and R₂, together, are a nitrogen protecting group or are joined ina ring structure that optionally includes an additional heteroatomselected from N and O; and

each R₃ and R₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protectinggroup, or R₃ and R₄, together, are a nitrogen protecting group;

or R₃ and R₄ are joined in a ring structure that optionally includes anadditional heteroatom selected from N and O.

In one embodiment R₁ is H, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl andR₂ is C₁-C₁₀ substituted alkyl. In another embodiment R₁ is C₁-C₁₀alkyl. In a further embodiment R₂ is C₁-C₁₀ substituted alkyl and thesubstituent is NH₃ ⁺ or N(R₃)(R₄). In another embodiment R₁ and R₂ areboth C₁-C₁₀ substituted alkyl with preferred substituents independentlyselected from NH₃ ⁺ and N(R₃)(R₄). In yet a further embodiment both R₁and R₂ are C₁-C₁₀ alkyl.

In one embodiment R₁ and R₂ are joined in a ring structure that caninclude at least one heteroatom selected from N and O. Preferred ringstructures are imidazole, piperidine, morpholine or a substitutedpiperazine with a preferred substituent being C₁-C₁₂ alkyl.

In one embodiment the heterocyclic base is a purine or a pyrimidine withpreferred heterocyclic bases being adenine, cytosine, 5-methylcytosine,thymine, uracil, guanine or 2-aminoadenine.

In one embodiment the oligomeric compound comprises from about 5 toabout 50 nucleosides. In a preferred embodiment the oligomeric compoundcomprises from about 8 to about 30 nucleosides with a preferred rangefrom about 15 to about 25 nucleosides.

The present invention also includes nucleosidic compounds of theformula:

wherein:

Bx is a heterocyclic base;

T₁ and T₂, independently, are OH, a protected hydroxyl, an activatedphosphorus group, a reactive group for forming an internucleotidelinkage, a nucleoside, a nucleotide, an oligonucleoside anoligonucleotide or a linkage to a solid support;

each R₁ and R₂ is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, whereinsaid substitution is OR₃, SR₃, NH₃ ⁺, N (R₃)(R₄), guanidino or acylwhere said acyl is an acid, amide or an ester;

or R₁ and R₂, together, are a nitrogen protecting group or are joined ina ring structure that optionally includes an additional heteroatomselected from N and O; and

each R₃ and R₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protectinggroup, or R₃ and R₄, together, are a nitrogen protecting group;

or R₃ and R₄ are joined in a ring structure that optionally includes anadditional heteroatom selected from N and O.

In one embodiment R₁ is H, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl andR₂ is C₁-C₁₀ substituted alkyl. In another embodiment R₁ is C₁-C₁₀alkyl. In a further embodiment R₂ is C₁-C₁₀ substituted alkyl and thesubstituent is NH₃ ⁺ or N (R₃)(R₄). In another embodiment R₁ and R₂ areboth C₁-C₁₀ substituted alkyl with preferred substituents independentlyselected from NH₃ ⁺ and N (R₃)(R₄). In yet a further embodiment both R₁and R₂ are C₁-C₁₀ alkyl.

In one embodiment R₁ and R₂ are joined in a ring structure that caninclude at least one heteroatom selected from N and O. Preferred ringstructures are imidazole, piperidine, morpholine or a substitutedpiperazine with a preferred substituent being C₁-C₁₂ alkyl.

In one embodiment the heterocyclic base is a purine or a pyrimidine withpreferred heterocyclic bases being adenine, cytosine, 5-methylcytosine,thymine, uracil, guanine or 2-aminoadenine.

In one embodiment T₁is a hydroxyl protecting group. In anotherembodiment T₂ is an activated phosphorus group or a connection to asolid support. A preferred solid support material is microparticles.With CPG being a more preferred solid support material.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel 2′-O-modified nucleosidicmonomers and to oligomeric compounds incorporating these novel2′-O-modified nucleosidic monomers. These modifications have certaindesirable properties that contribute toward increases in bindingaffinity and/or nuclease resistance.

There are a number of items to consider when designing oligomericcompounds having enhanced binding affinities. One effective approach toconstructing oligomeric compounds with very high RNA binding affinityrelates to the combination of two or more different types ofmodifications, each of which contributes favorably to various factorsthat might be important for binding affinity.

Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443,recently published a study on the influence of structural modificationsof oligonucleotides on the stability of their duplexes with target RNA.In this study, the authors reviewed a series of oligonucleotidescontaining more than 200 different modifications that had beensynthesized and assessed for their hybridization affinity and T_(m).Sugar modifications studied included substitutions on the 2′-position ofthe sugar, 3′-substitution, replacement of the 4′-oxygen, the use ofbicyclic sugars, and four member ring replacements. Several heterocyclicbase modifications were also studied including substitutions at the 5,or 6 position of thymine, modifications of pyrimidine heterocycles andmodifications of purine heterocycles. Numerous backbone modificationswere also investigated including backbones bearing phosphorus, backbonesthat did not bear a phosphorus atom, and backbones that were neutral.

Four general approaches potentially may be used to improve hybridizationof oligonucleotides to RNA targets. These include: preorganization ofthe sugars and phosphates of the oligodeoxynucleotide strand intoconformations favorable for hybrid formation, improving stacking ofnucleobases by the addition of polarizable groups to the heterocyclebases of the nucleosidic monomers of the oligonucleotide, increasing thenumber of H-bonds available for A-U pairing, and neutralization ofbackbone charge to facilitate removing undesirable repulsiveinteractions. This invention principally employs the first of these,preorganization of the sugars and phosphates of the oligodeoxynucleotidestrand into conformations favorable for hybrid formation, and can beused in combination with the other three approaches.

Sugars in DNA:RNA hybrid duplexes frequently adopt a C3′ endoconformation. Thus, modifications that shift the conformationalequilibrium of the sugar moieties in the single strand toward thisconformation should preorganize the antisense strand for binding to RNA.Of the several sugar modifications that have been reported and studiedin the literature, the incorporation of electronegative substituentssuch as 2′-fluoro or 2′-alkoxy shift the sugar conformation towards the3′ endo (northern) pucker conformation. This pucker conformation furtherassisted in increasing the Tm of the oligonucleotide with its target.

There is a clear correlation between substituent size at the 2′-positionand duplex stability. Incorporation of alkyl substituents at the2′-position typically leads to a significant decrease in bindingaffinity. Thus, small alkoxy groups generally are very favorable whilelarger alkoxy groups at the 2′-position generally are unfavorable.However, if the 2′-substituent contained an ethylene glycol motif, thena strong improvement in binding affinity to the target RNA is observed.

The high binding affinity resulting from 2′-substitution has beenpartially attributed to the 2′-substitution causing a C3′ endo sugarpucker which in turn may give the oligomer a favorable A-form likegeometry. This is a reasonable hypothesis since substitution at the 2′position by a variety of electronegative groups (such as fluoro andO-alkyl chains) has been demonstrated to cause C3′ endo sugar puckering(De Mesmaeker et al., Acc. Chem. Res., 1995, 28, 366-374; Lesnik et al.,Biochemistry, 1993, 32, 7832-7838).

In addition, for 2′-substituents containing an ethylene glycol motif, agauche interaction between the oxygen atoms around the O—C—C—O torsionof the side chain may have a stabilizing effect on the duplex (Freier etal., Nucleic Acids Research, (1997) 25:4429-4442). Such gaucheinteractions have been observed experimentally for a number of years(Wolfe et al., Acc. Chem. Res., 1972, 5, 102; Abe et al., J. Am. Chem.Soc., 1976, 98, 468). This gauche effect may result in a configurationof the side chain that is favorable for duplex formation. The exactnature of this stabilizing configuration has not yet been explained.While we do not want to be bound by theory, it may be that holding theO—C—C—O torsion in a single gauche configuration, rather than a morerandom distribution seen in an alkyl side chain, provides an entropicadvantage for duplex formation.

The present invention has 2′ side chain having the formula:2′—OCH₂CH₂OCH₂CH₂N (R₁)(R₂), where R₁ and R₂ can each be alkyl orsubstituted alkyl groups which gives a tertiary amine capable of beingprotonated. When R₁ and R₂ are both methyl groups the pKa of the sidechain is 9.0 to 10.0 (aliphatic saturated 3° amine). This tertiary amineis expected to be protonated at physiological pH (7.0), and in endosomesand lysosomes (pH 5.0). The resulting positive charge should improve thebiostability of the drug by either inhibiting the nuclease from bindingto the oligonucleotide or displacing the metal ions needed for thenucleases to carry on their function (Beese et al., EMBO J., 1991, 10,25-33; and Brautigam et al., J. Mol. Bio., 1998, 277, 363-377).

As used herein, the term oligonucleoside includes oligomers or polymerscontaining two or more nucleoside subunits having a non-phosphorouslinking moiety. Oligonucleosides according to the invention aremonomeric subunits having a ribofuranose moiety attached to aheterocyclic base via a glycosyl bond. An oligonucleotide/nucleoside forthe purposes of the present invention is a mixed backbone oligomerhaving at least two nucleosides covalently bound by a non-phosphatelinkage and at least one phosphorous containing covalent bond with anucleotide, and wherein at least one of the monomeric nucleotide ornucleoside units is a 2′-O-substituted compound prepared using theprocess of the present invention. An oligo-nucleotide/nucleoside canadditionally have a plurality of nucleotides and nucleosides coupledthrough phosphorous containing and/or non-phosphorous containinglinkages.

In the context of this invention, the term “oligomeric compound” refersto a plurality of nucleosides joined together in a specific sequencefrom naturally and non-naturally occurring nucleosides. The termincludes oligonucleotides, oligonucleotide analogs, oligonucleosideshaving non-phosphorus containing internucleoside linkages and chimericoligomeric compounds having mixed internucleoside linkages which caninclude all phosphorus or phosphorus and non-phosphorus containinginternucleoside linkages. Each of the oligomeric compounds of theinvention have at least one modified nucleoside where the modificationis an aminooxy compound of the invention. Preferred nucleosides of theinvention are joined through a sugar moiety via phosphorus linkages, andinclude adenine, guanine, adenine, cytosine, uracil, thymine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyladenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosineand 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8-substituted guanines, other aza and deaza uracils, other aza anddeaza thymidines, other aza and deaza cytosines, other aza and deazaadenines, other aza and deaza guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

phosphorus containing linkages

phosphorodithioate (—O—P(S)(S)—O—);

phosphorothioate (—O—P(S)(O)—O—);

phosphoramidate (—O—P(O)(NJ)—O—);

phosphonate (—O—P(J)(O)—O—);

phosphotriesters (—O—P(O J)(O)—O—);

phophosphoramidate (—O—P(O)(NJ)—S—);

thionoalkylphosphonate (—O—P(S)(J)—O—);

thionoalkylphosphotriester (—O—P(O)(OJ)—S—);

boranophosphate (—R⁵—P(O)(O)—J—);

non-phosphorus containing linkages

thiodiester (—O—C(O)—S—);

thionocarbamate (—O—C(O)(NJ)—S—);

siloxane (—O—Si(J)₂—O—);

carbamate (—O—C(O)—NH— and —NH—C(O)—O—)

sulfamate (—C—S(O)(O)—N— and —N—S(O)(O)—N—;

morpholino sulfamide (—O—S(O)(N(morpholino)—);

sulfonamide (—O—SO₂—NH—);

sulfide (—CH₂—S—CH₂—);

sulfonate (—O—SO₂—CH₂—);

N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—);

thioformacetal (—S—CH₂—O—);

formacetal (—O—CH₂—O—);

thioketal (—S—C(J)₂—O—); and

ketal (—O—C(J)₂—O—);

amine (—NH—CH₂—CH₂—);

hydroxylamine (—CH₂—N(J)—O—);

hydroxylimine (—CH═N—O—); and

hydrazinyl (—CH₂—N(H)—N(H)—).

where“J” denotes a substituent group which is commonly hydrogen or analkyl group or a more complicated group that varies from one type oflinkage to another.

In addition to linking groups as described above that involve themodification or substitution of the —O—P—O— atoms of a naturallyoccurring linkage, included within the scope of the present inventionare linking groups that include modification of the 5′-methylene groupas well as one or more of the —O—P—O— atoms. Linkages of this type arewell documented in the prior art and include without limitation thefollowing:

amides (—CH₂—CH₂—N(H)—C(O)) and —CH₂—O—N═CH—; and

alkylphosphorus (—C(J)₂—P(═O)(OJ)—C(J)₂—C(J)₂—).

wherein J is as described above.

Synthetic schemes for the synthesis of the substitute internucleosidelinkages described above are disclosed in: WO 91/08213; WO 90/15065; WO91/15500; WO 92/20822; WO 92/20823; WO 91/15500; WO 89/12060; EP 216860;U.S. Pat. Nos. 9,204,294; 9,003,138; 9,106,855; 9,203,385; 9,103,680;U.S. Pat. Ser. Nos. 07/990,848; 07/892,902; 07/806,710; 07/763,130;07/690,786; U.S. Pat. Nos. 5,466,677; 5,034,506; 5,124,047; 5,278,302;5,321,131; 5,519,126; 4,469,863; 5,455,233; 5,214,134; 5,470,967;5,434,257; Stirchak, E. P., et al., Nucleic Acid Res., 1989, 17,6129-6141; Hewitt, J. M., et al., 1992, 11, 1661-1666; Sood, A., et al.,J. Am. Chem. Soc., 1990, 112, 9000-9001; Vaseur, J. J. et al., J. Amer.Chem. Soc., 1992, 114, 4006-4007; Musichi, B., et al., J. Org. Chem.,1990, 55, 4231-4233; Reynolds, R. C., et al., J. Org. Chem., 1992, 57,2983-2985; Mertes, M. P., et al., J. Med. Chem., 1969, 12, 154-157;Mungall, W. S., et al., J. Org. Chem., 1977, 42, 703-706; Stirchak, E.P., et al., J. Org. Chem., 1987, 52, 4202-4206; Coull, J. M., et al.,Tet. Lett., 1987, 28, 745; and Wang, H., et al., Tet. Lett., 1991, 32,7385-7388.

The nucleosidic monomers and oligomeric compounds of the invention caninclude modified sugars and modified bases (see, e.g., U.S. Pat. No.3,687,808 and PCT application PCT/US89/02323). Such oligomeric compoundsare best described as being structurally distinguishable from, yetfunctionally interchangeable with, naturally occurring or synthetic wildtype oligonucleotides. Representative modified sugars includecarbocyclic or acyclic sugars, sugars having substituent groups at their2′ position, sugars having substituent groups at their 3′ position, andsugars having substituents in place of one or more hydrogen atoms of thesugar. Representative modifications are disclosed in InternationalPublication Numbers WO 91/10671, published Jul. 25, 1991, WO 92/02258,published Feb. 20, 1992, WO 92/03568, published Mar. 5, 1992, and U.S.Pat. Nos. 5,138,045, 5,218,105, 5,223,618 5,359,044, 5,378,825,5,386,023, 5,457,191, 5,459,255, 5,489,677, 5,506,351, 5,541,307,5,543,507, 5,571,902, 5,578,718, 5,587,361, 5,587,469, all assigned tothe assignee of this application. The disclosures of each of the abovereferenced publications are herein incorporated by reference.

Additional modifications may also be made at for example the 3′ positionof the sugar on the 3′ terminal nucleosidic monomer and the 5′ positionof the 5′ terminal nucleosidic monomer. In one aspect of the inventionmoieties or conjugates which enhance activity, cellular distribution orcellular uptake are chemically linked to one or more positions that areavailable for modification. Such moieties include but are not limited tolipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al.,Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & nucleosidic monomers, 1995, 14, 969), or adamantane aceticacid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmitylmoiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Representative heterocyclic bases amenable to the present inventioninclude guanine, cytosine, uridine, and thymine, as well as othersynthetic and natural nucleobases such as xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl,hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine. Further purines and pyrimidines include those disclosedin U.S. Pat. No. 3,687,808, those disclosed in the Concise EncyclopediaOf Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I.,ed. John Wiley & Sons, 1990, and those disclosed by Englisch, et al.,Angewandte Chemie, International Edition 1991, 30, 613. All sucholigomeric compounds are comprehended by this invention.

The nucleosidic monomers used in preparing oligomeric compounds of thepresent invention can include appropriate activated phosphorus groupssuch as activated phosphate groups and activated phosphite groups. Asused herein, the terms activated phosphate and activated phosphitegroups refer to activated monomers or oligomers that are reactive with ahydroxyl group of another monomeric or oligomeric compound to form aphosphorus-containing internucleotide linkage. Such activated phosphorusgroups contain activated phosphorus atoms in p^(III) or p^(V) valencystates. Such activated phosphorus atoms are known in the art andinclude, but are not limited to, phosphoramdite, H-phosphonate andphosphate triesters. A preferred synthetic solid phase synthesisutilizes phosphoramidites as activated phosphates. The phosphoramiditesutilize P^(III) chemistry. The intermediate phosphite compounds aresubsequently oxidized to the P^(V) state using known methods to yield,in a preferred embodiment, phosphodiester or phosphorothioateinternucleotide linkages. Additional activated phosphates and phosphitesare disclosed in Tetrahedron Report Number 309 (Beaucage and Iyer,Tetrahedron, 1992, 48, 2223-2311).

A number of chemical functional groups can be introduced into compoundsof the invention in a blocked form and subsequently deblocked to form afinal, desired compound. Such groups can be introduced as groupsdirectly or indirectly attached at the heterocyclic base and the sugarsubstituents at the 2′, 3′ and 5′-positions. In general, a blockinggroup renders a chemical functionality of a larger molecule inert tospecific reaction conditions and can later be removed from suchfunctionality without substantially damaging the remainder of themolecule (Green and Wuts, Protective Groups in Organic Synthesis, 2dedition, John Wiley & Sons, New York, 1991). For example, the nitrogenatom of amino groups can be blocked as phthalimido groups, as9-fluorenylmethoxycarbonyl (FMOC) groups, and withtriphenylmethylsulfenyl, t-BOC or benzyl groups. Carboxyl groups can beprotected as acetyl groups. Representative hydroxyl protecting groupsare described by Beaucage et al., Tetrahedron 1992, 48, 2223. Preferredhydroxyl protecting groups are acid-labile, such as the trityl,monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl(MOX). Chemical functional groups can also be “blocked” by includingthem in a precursor form. Thus, an azido group can be used considered asa “blocked” form of an amine since the azido group is easily convertedto the amine. Further representative protecting groups utilized inoligonucleotide synthesis are discussed in Agrawal, et al., Protocolsfor Oligonucleotide Conjugates, Eds, Humana Press; New Jersey, 1994;Vol. 26 pp. 1-72.

Examples of hydroxyl protecting groups include, but are not limited to,t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl,diphenylmethyl, p,p′-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate,chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate,p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.

Amino-protecting groups stable to acid treatment are selectively removedwith base treatment, and are used to make reactive amino groupsselectively available for substitution. Examples of such groups are theFmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J.Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p.1), andvarious substituted sulfonylethyl carbamates exemplified by the Nscgroup (Samukov et al.,Tetrahedron Lett, 1994, 35:7821; Verhart andTesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).

Additional amino-protecting groups include but are not limited to,carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; andimine- and cyclic imide-protecting groups, such as phthalimido anddithiasuccinoyl. Equivalents of these amino-protecting groups are alsoencompassed by the compounds and methods of the present invention.

In some especially preferred embodiments, one or more of theinternucleoside linkages comprising oligomeric compounds of theinvention are optionally protected phosphorothioate internucleosidelinkages. Representative protecting groups for phosphorus containinginternucleoside linkages such as phosphite, phosphodiester andphosphoro-thioate linkages include β-cyanoethyl, diphenylsilylethyl,δ-cyanobutenyl, cyano p-xylyl (CPX), N-methyl-N-trifluoro-acetyl ethyl(META), acetoxy phenoxy ethyl (APE) and butene-4-yl groups. See forexample U.S. Pat. No. 4,725,677 and U.S. Pat. No. Re. 34,069(β-cyanoethyl); Beaucage, S. L. and Iyer, R. P., Tetrahedron, 49 No. 10,pp. 1925-1963 (1993); Beaucage, S. L. and Iyer, R. P., Tetrahedron, 49No. 46, pp. 10441-10488 (1993); Beaucage, S. L. and Iyer, R. P.,Tetrahedron, 48 No. 12, pp. 2223-2311 (1992).

In the context of this specification, alkyl (generally C₁-C₂₀), alkenyl(generally C₂-C₂₀), and alkynyl (generally C₂-C₂₀) (with more preferredranges from C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl and C₂-C₁₀ alkynyl), groupsinclude but are not limited to substituted and unsubstituted straightchain, branch chain, and alicyclic hydrocarbons, including methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, nonadecyl, eicosyl and other higher carbon alkylgroups. Further examples include 2-methylpropyl, 2-methyl-4-ethylbutyl,2,4-diethylbutyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl,6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl,2-ethylhexyl and other branched chain groups, allyl, crotyl, propargyl,2-pentenyl and other unsaturated groups containing a pi bond,cyclohexane, cyclopentane, adamantane as well as other alicyclic groups,3-penten-2-one, 3-methyl-2-butanol, 2-cyanooctyl, 3-methoxy-4-heptanal,3-nitrobutyl, 4-isopropoxydodecyl, 4-azido-2-nitrodecyl,5-mercaptononyl, 4-amino-1-pentenyl as well as other substituted groups.Representative alkyl substituents are disclosed in U.S. Pat. No.5,212,295, at column 12, lines 41-50, hereby incorporated by referencein its entirety.

Further, in the context of this invention, a straight chain compoundmeans an open chain compound, such as an aliphatic compound, includingalkyl, alkenyl, or alkynyl compounds; lower alkyl, alkenyl, or alkynylas used herein include but are not limited to hydrocarbyl compounds fromabout 1 to about 6 carbon atoms. A branched compound, as used herein,comprises a straight chain compound, such as an alkyl, alkenyl, alkynylcompound, which has further straight or branched chains attached to thecarbon atoms of the straight chain.

A cyclic compound, as used herein, refers to closed chain compounds,i.e. a ring of carbon atoms, such as an alicyclic or aromatic compound.The straight, branched, or cyclic compounds may be internallyinterrupted, as in alkoxy or heterocyclic compounds. In the context ofthis invention, internally interrupted means that the carbon chains maybe interrupted with heteroatoms such as O, N, or S. However, if desired,the carbon chain may have no heteroatoms.

Compounds of the invention can include ring structures that include anitrogen atom (e.g., —N(R₁) (R₂) and —N(R₃) (R₄) where (R₁) (R₂) and(R₃) (R₄) each form cyclic structures about the respective N to whichthey are attached). The resulting ring structure is a heterocycle or aheterocyclic ring structure that can include further heteroatomsselected from N, O and S. Such ring structures may be mono-, bi- ortricyclic, and may be substituted with substituents such as oxo, acyl,alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido, azido,aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, haloalkyl,haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, sulfone,sulfonamide, thiol and thioalkoxy. A preferred bicyclic ring structurethat includes nitrogen is phthalimido.

In general, the term “hetero” denotes an atom other than carbon,preferably but not exclusively N, O, or S. Accordingly, the term“heterocyclic ring” denotes an alkyl ring system having one or moreheteroatoms (i.e., non-carbon atoms). Heterocyclic ring structures ofthe present invention can be fully saturated, partially saturated,unsaturated or with a polycyclic heterocyclic ring each of the rings maybe in any of the available states of saturation. Heterocyclic ringstructures of the present invention also include heteroaryl whichincludes fused systems including systems where one or more of the fusedrings contain no heteroatoms. Heterocycles, including nitrogenheterocycles, according to the present invention include, but are notlimited to, imidazole, pyrrole, pyrazole, indole, 1H-indazole,α-carboline, carbazole, phenothiazine, phenoxazine, tetrazole, triazole,pyrrolidine, piperidine, piperazine and morpholine groups. A morepreferred group of nitrogen heterocycles includes imidazole, pyrrole,indole, and carbazole groups.

In the context of this specification, aryl groups are substituted andunsubstituted aromatic cyclic moieties including but not limited tophenyl, naphthyl, anthracyl, phenanthryl, pyrenyl, and xylyl groups.Alkaryl groups are those in which an aryl moiety links an alkyl moietyto a core structure, and aralkyl groups are those in which an alkylmoiety links an aryl moiety to a core structure.

Oligomeric compounds according to the present invention that arehybridizable to a target nucleic acid preferably comprise from about 5to about 50 nucleosides. It is more preferred that such compoundscomprise from about 8 to about 30 nucleosides, with 15 to 25 nucleosidesbeing particularly preferred. As used herein, a target nucleic acid isany nucleic acid that can hybridize with a complementary nucleicacid-like compound. Further in the context of this invention,“hybridization” shall mean hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding between complementarynucleobases. “Complementary” as used herein, refers to the capacity forprecise pairing between two nucleobases. For example, adenine andthymine are complementary nucleobases which pair through the formationof hydrogen bonds. “Complementary” and “specifically hybridizable,” asused herein, refer to precise pairing or sequence complementaritybetween a first and a second nucleic acid-like oligomers containingnucleoside subunits. For example, if a nucleobase at a certain positionof the first nucleic acid is capable of hydrogen bonding with anucleobase at the same position of the second nucleic acid, then thefirst nucleic acid and the second nucleic acid are considered to becomplementary to each other at that position. The first and secondnucleic acids are complementary to each other when a sufficient numberof corresponding positions in each molecule are occupied by nucleobaseswhich can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between a compound of the invention and a target RNAmolecule.

It is understood that an oligomeric compound of the invention need notbe 100% complementary to its target RNA sequence to be specificallyhybridizable. An oligomeric compound is specifically hybridizable whenbinding of the oligomeric compound to the target RNA molecule interfereswith the normal function of the target RNA to cause a loss of utility,and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligomeric compound to non-target sequencesunder conditions in which specific binding is desired, i.e. underphysiological conditions in the case of in vivo assays or therapeutictreatment, or in the case of in vitro assays, under conditions in whichthe assays are performed.

The oligomeric compounds of the present invention can be used indiagnostics, therapeutics and as research reagents. They can be used inpharmaceutical compositions by including a suitable pharmaceuticallyacceptable diluent or carrier. They further can be used for treatingorganisms having a disease characterized by the undesired production ofa protein. The organism should be contacted with an oligonucleotidehaving a sequence that is capable of specifically hybridizing with astrand of nucleic acid coding for the undesirable protein. Treatments ofthis type can be practiced on a variety of organisms ranging fromunicellular prokaryotic and eukaryotic organisms to multicellulareukaryotic organisms. Any organism that utilizes RNA-DNA transcriptionor RNA-protein translation as a fundamental part of its hereditary,metabolic or cellular control is susceptible to therapeutic and/orprophylactic treatment in accordance with this invention. Seeminglydiverse organisms such as bacteria, yeast, protozoa, algae, all plantsand all higher animal forms including warm-blooded animals, ca betreated. Further each cell of multicellular eukaryotes can be treatedsince they include both DNA-RNA transcription and RNA-proteintranslation as integral parts of their cellular activity. Many of theorganelles (e.g., mitochondria and chloroplasts) of eukaryotic cellsalso include transcription and translation mechanisms. Thus, singlecells, cellular populations or organelles can also be included withinthe definition of organisms that can be treated with therapeutic ordiagnostic oligomeric compounds. As used herein, therapeutics is meantto include the eradication of a disease state, by killing an organism orby control of erratic or harmful cellular growth or expression.

Oligomeric compounds according to the invention can be assembled insolution or through solid-phase reactions, for example, on a suitableDNA synthesizer utilizing nucleosides, phosphoramidites and derivatizedcontrolled pore glass (CPG) according to the invention and/or standardnucleosidic monomer precursors. In addition to nucleosides that includea novel modification of the inventions other nucleoside within anoligonucleotide may be further modified with other modifications at the2′ position. Precursor nucleoside and nucleosidic monomer precursorsused to form such additional modification may carry substituents eitherat the 2′ or 3′ positions. Such precursors may be synthesized accordingto the present invention by reacting appropriately protected nucleosidesbearing at least one free 2′ or 3′ hydroxyl group with an appropriatealkylating agent such as, but not limited to, alkoxyalkyl halides,alkoxylalkylsulfonates, hydroxyalkyl halides, hydroxyalkyl sulfonates,aminoalkyl halides, aminoalkyl sulfonates, phthalimidoalkyl halides,phthalimidoalkyl sulfonates, alkylaminoalkyl halides, alkylaminoalkylsulfonates, dialkylaminoalkyl halides, dialkylaminoalkylsulfonates,dialkylaminooxyalkyl halides, dialkylaminooxyalkyl sulfonates andsuitably protected versions of the same. Preferred halides used foralkylating reactions include chloride, bromide, fluoride and iodide.Preferred sulfonate leaving groups used for alkylating reactionsinclude, but are not limited to, benzenesulfonate, methylsulfonate,tosylate, p-bromobenzenesulfonate, triflate, trifluoroethylsulfonate,and (2,4-dinitroanilino)-benzenesulfonate.

Suitably protected nucleosides can be assembled into oligomericcompounds according to known techniques. See, for example, Beaucage etal., Tetrahedron, 1992, 48, 2223.

The ability of oligomeric compounds to bind to their complementarytarget strands is compared by determining the melting temperature(T_(m)) of the hybridization complex of the oligonucleotide and itscomplementary strand. The melting temperature (T_(m)), a characteristicphysical property of double helices, denotes the temperature (in degreescentigrade) at which 50% helical (hybridized) versus coil (unhybridized)forms are present. T_(m) is measured by using the UV spectrum todetermine the formation and breakdown (melting) of the hybridizationcomplex. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently, a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m), the greater the strength of the bonds between thestrands. The structure-stability relationships of a large number ofnucleic acid modifications have been reviewed (Freier and Altmann, Nucl.Acids Research, 1997, 25, 4429-443).

The relative binding ability of the oligomeric compounds of the presentinvention was determined using protocols as described in the literature(Freier and Altmann, Nucl. Acids Research, 1997, 25, 4429-443).Typically absorbance versus temperature curves were determined usingsamples containing 4 uM oligonucleotide in 100 mM Na+, 10 mM phosphate,0.1 mM EDTA, and 4 uM complementary, length matched RNA.

The in vivo stability of oligomeric compounds is an important factor toconsider in the development of oligonucleotide therapeutics. Resistanceof oligomeric compounds to degradation by nucleases, phosphodiesterasesand other enzymes is therefore determined. Typical in vivo assessment ofstability of the oligomeric compounds of the present invention isperformed by administering a single dose of 5 mg/kg of oligonucleotidein phosphate buffered saline to BALB/c mice. Blood collected at specifictime intervals post-administration is analyzed by HPLC or capillary gelelectrophoresis (CGE) to determine the amount of the oligomeric compoundremaining intact in circulation and the nature the of the degradationproducts.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples, which are not intended to be limiting. Alloligonucleotide sequences are listed in a standard 5′ to 3′ order fromleft to right.

EXAMPLE 1 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) was slowlyadded to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolved as the soliddissolved O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) were added and the bomb was sealed, placed in anoil bath and heated to 155° C. for 26hours. The bomb was cooled to roomtemperature and opened. The crude solution was concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess alcohol was extracted into the hexane layer. The aqueous layerwas extracted with ethyl acetate (3×200 mL) and the combined organiclayers were washed once with water, dried over anhydrous sodium sulfateand concentrated. The residue was columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions were concentrated a colorless solidformed which was collected to give 490 mg of the title compound as awhite solid. Rf=0.56 in CH₂—CH₁₂:CH₃—OH (10:1); MS/ES calculated 374;observed 374.5.

EXAMPLE 25′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) were added and stirred for 1 hour. The reactionmixture was poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers were washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gave 0.72 g (82%) of the title compound.

EXAMPLE 35′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl) phosphoramidate

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) were added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture was stirred overnight and the solventevaporated. The resulting residue was purified by silica gel flashcolumn chromatography with ethyl acetate as the eluent to give 1.98 g(83% yield) of the title compound.

EXAMPLE 45′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl-uridine-3′-O-succinate

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyluridine(270 mg, 0.41 mmol) was heated with 68 mg of succinic anhydride (0.6mmol), 4-N,N-dimethylamino pyridine (24 mg) and Et₃N (56 μL) indichloroethane (1 mL) at 50° C. for 10 minutes in a Pyrex tube in aheating block. After cooling, the reaction mixture was diluted withmethylene chloride (20 mL) and washed with a 10% aqueous solution ofcitric acid (3×20 mL) followed by water. The resulting solution wasdried over anhydrous Na₂SO₄ to give 217 mg (58% yield) of the titlecompound.

TLC indicated (CH₂Cl₂/MeOH, 10:1) a polar product at the origin, asexpected.

EXAMPLE 5 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl-uridine-3′-O-succinylcontrolled pore glass (CPG)

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl-uridine-3′-O-succinate(116 mg, 0.15 mmol, 2 eq.) was dried under high vacuum overnight. Tothis dried material was added CPG (650 mg, 1 eq.), anhydrous DMF (2 mL),N-methylmorpholine (33 μL, 4 eq.) and 2-1H-benzotriazole-1-yl2-1H-benzotriazole-1-yl-1,1,3,3-tetramethyluronium-tetrafluoro-borate(TBTU, 48 mg, 2 eq.) was added to the reaction mixture with shaking for12 hours. The CPG was then filtered and washed with DMF, CH₂Cl₂, CH₃CNand Et₂O. Finally, it was dried and capped with acetic anhydride/Et₃N.The loading of the CPG was determined via the dimethoxytrityl assaymethod to be 53 μmoles/g.

EXAMPLE 6 2-[2-(dimethylamino)ethylmercapto]ethanol

2-(Dimethylamino)ethanethiol hydrochloride (Aldrich) is treated withNaOH (0.2N) in ethanol (95%). To this slurry, 2-bromoethanol (1.2 eq.)is added and the mixture is refluxed for 2 hours. The reaction mixtureis cooled, filtered and concentrated. The resultant residue is purifiedby silica gel flash column chromatography to give the title compound.

EXAMPLE 7 2′-O-[2-[2-((dimethylamino)ethyl)mercapto]ethyl]-5-methyluridine

2-[2-((dimethylamino)ethyl)mercapto] ethanol (50 mmol) is slowly addedto a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol) withstirring in a 100 mL bomb. Hydrogen gas is evolved as the soliddissolves. O², 2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, then placed in anoil bath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into hexanes. The aqueous layer is extractedwith ethyl acetate (3×200 mL) and the combined organic layer is washedonce with water and dried over anhydrous sodium sulfate andconcentrated. The resultant residue is purified by silica gel flashcolumn chromatography using methanol/methylene chloride having 2%triethylamine to give the title compound.

EXAMPLE 85′-O-Dimethoxytrityl-2′-O-2-[2-((dimethylamino)ethyl)mercapto]ethyl)]5-methyluridine

To 2′-O-2-[2-((dimethylamino)ethyl)mercapto]ethyl)]5-methyl uridine (1.3mmol) in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and DMT-Cl(0.87 g, 2 eq.) are added and stirred for 1 hour. The reaction mixtureis poured into water (200 mL) and extracted with CH₂Cl₂ (2×200 mL). Thecombined CH₂Cl₂ layers are washed with saturated NaHCO₃ solution,saturated NaCl solution, dried over anhydrous sodium sulfate andconcentrated. The resultant residue is purified by silica gel flashcolumn chromatography using methanol/methylene chloride having 1%triethylamine to give the title compound.

EXAMPLE 95′-O-Dimethoxytrityl-2′-O-2-[2-((dimethylamino)ethyl)mercapto]ethyl)]5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl) phosphoramidate

5′-O-Dimethoxytrityl-2′-O-2-[2-((dimethylamino)ethyl)mercapto]ethyl)]5-methyluridine(3 mmol) is dissolved in CH₂Cl₂ (20 mL) and to this solution, underargon, diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are added.The reaction is stirred overnight and the solvent is evaporated. Theresultant residue is purified by silica gel flash column chromatographyusing ethyl acetate to give the title phosphoramidite.

EXAMPLE 105′-O-Dimethoxytrityl-2′-O-2-[2-((dimethylamino)ethyl)mercapto]ethyl)]5-methyl-uridine-3′-O-succinate

5′-O-Dimethoxytrityl-2′-O-2-[2-((dimethylamino)ethyl)mercapto]ethyl)]5-methyluridine(0.41 mmol) is heated with succinic anhydride (68 mg, 0.6 mmol),4-N,N-dimethylamino pyridine (24 mg) and Et₃N (56 μL) in dichloroethane(1 mL) at 50° C. for 10 minutes in a Pyrex tube in a heating block.After cooling the reaction mixture is diluted with methylene chloride(20 mL) and washed with 10% citric acid aqueous solution (3×20 mL)followed by water and dried over anhydrous Na₂SO₄ to give the titlesuccinate.

EXAMPLE 115′-O-Dimethoxytrityl-2′-O-2-[2-((dimethylamino)ethyl)mercapto]ethyl)]5-methyl-uridine-3′-O-succinylcontrolled pore glass (CPG)

The succinate from Example 10 above (0.15 mmol, 2 eq.) is dried undervacuum overnight. CPG (650 mg, 1 eq.), anhydrous DMF (2 mL), 33 μL ofN-methylmorpholine (4 eq.) and 48 mg (2 eq.) of TBTU(2-1H-benzotriazole-1-yl) are added to the dried succinate.1,1,3,3-tetramethyluronium-tetrafluoroborate is added and the mixture isshaken for 12 hours. The CPG is then filtered and washed with DMF,CH₂Cl₂, CH₃CN and Et₂O. The CPG is dried and capped with aceticanhydride/Et₃N. The loading is determined using the standarddimethoxytrityl assay.

EXAMPLE 12 2-[2-(diethylamino)ethoxy] ethanol

2-(2-aminoethoxy)ethanol (Aldrich 0.5 mmol) is treated with NaBH₃CN(Aldrich, 200 mg, 3.0 mmol) in 50% aqueous methanol (30 mL). To thissolution, acetaldehyde 95% purity (2 mL, 17 mmol) is added in oneportion and the mixture is heated at 50° C. for 2 days in a flask underargon. After removal of the solvent under reduced pressure, the residueis dissolved in water, extracted with ethylacetate to give the titlecompound.

EXAMPLE 13 2′-O-[2(2-N,N-diethylaminoethoxy)ethyl]-5-methyl uridine

2[2-(Diethylamino)ethoxy]ethanol (50 mmol) is slowly added to a solutionof borane in tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a100 mL bomb. Hydrogen gas evolves as the solid dissolvesO²-2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate(2.5 mg) are added and the bomb is sealed, placed in an oil bath andheated to 155° C. for 26 hours. The bomb is cooled to room temperatureand opened. The crude solution is concentrated and the residuepartitioned between water (200 mL) and hexanes (200 mL). The excessalcohol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. The appropriate column fractions are concentrated andconcentrated to give the title compound.

EXAMPLE 145′-O-dimethoxytrityl-2′-O-[2(2-N,N-diethylaminoethoxy)ethyl)]-5-methyluridine

To 1.3 mmol of 2′-O-[2(2-N,N-diethylaminoethoxy)ethyl)]-5-methyl uridinein anhydrous pyridine (8 mL), triethylamine (0.36 mL) anddimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and stirredfor 1 hour. The reaction mixture is poured into water (200 mL) andextracted with CH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers are washedwith saturated NaHCO₃ solution, followed by saturated NaCl solution anddried over anhydrous sodium sulfate. Evaporation of the solvent followedby silica gel chromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1%triethylamine) gives the title compound.

EXAMPLE 155′-O-Dimethoxytrityl-2′-O-[2(2-N,N-diethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidate

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-diethylaminoethoxy)ethyl)]-5-methyluridine(3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere of argon. Thereaction mixture is stirred overnight and the solvent evaporated. Theresulting residue is purified by silica gel flash column chromatographywith ethyl acetate as the eluent to give title compound.

EXAMPLE 165′-O-Dimethoxytrityl-2′-O-[2(2-N,N-diethylaminoethoxy)ethyl)]-5-methyl-uridine-3′-O-succinate

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-diethylaminoethoxy)ethyl)]-5-methyluridine(0.41 mmol) is heated with 68 mg of succinic anhydride (0.6 mmol),4-N,N-diethylamino pyridine (24 mg) and Et₃N (56 μL) in dichloroethane(1 mL) at 50° C. for 10 minutes in a Pyrex tube in a heating block.After cooling, the reaction mixture is diluted with methylene chloride(20 mL) and washed with a 10% aqueous solution of citric acid (3×20 mL)followed by water. The resulting solution is dried over anhydrous Na₂SO₄to give the title compound.

EXAMPLE 175′-O-Dimethoxytrityl-2′-O-[2(2-N,N-diethylaminoethoxy)ethyl)]-5-methyl-uridine-3′-O-succinylcontrolled pore glass (CPG)

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-diethylaminoethoxy)ethyl)]-5-methyl-uridine-3′-O-succinate(0.15 mmol, 2 eq.) is dried under high vacuum overnight. To this driedmaterial is added CPG (650 mg, 1 eq.), anhydrous DMF (2 mL),N-methylmorpholine (33 μL, 4 eq.) and 2-1H-benzotriazole-1-yl-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU, 48 mg, 2 eq.) isadded to the reaction mixture with shaking for 12 hours. The CPG is thenfiltered and washed with DMF, CH₂Cl₂, CH₃CN and Et₂O. Finally, it isdried and capped with acetic anhydride/Et₃N. The loading of the CPG isdetermined via the dimethoxytrityl assay method.

EXAMPLE 18 2-[bis-2-(N,N-dimethylamino-ethyl)amino ethoxy] ethanol

N,N-Dimethylaminoacetaldehyde diethyl acetal (Aldrich, 1 mmol) istreated with aqueous solution of trifluoroacetic acid and refluxedovernight to give N,N-dimethylamino acetaldehyde.2-(2-Aminoethoxy)ethanol is treated with NaBH₃CN and N,N-dimethylaminoacetaldehyde in methanol solvent and refluxed over night. After removalof the solvent under reduced pressure, the residue is dissolved inwater, extracted with ethyl acetate and purified by columnchromatography to give the title compound.

EXAMPLE 19 2′-O-[2(bis-2-N,N-dimethylaminoethyl)ethoxy)ethyl]-5-methyluridine

2[2-(Bis-N,N-dimethylaminomethyl)ethoxy]ethanol (50 mmol) is slowlyadded to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves O²-2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess alcohol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. The column fractions are concentrated to give the titlecompound.

EXAMPLE 205′-O-Dimethoxytrityl-2′-O-[2(2-(bis-N,N-dimethylaminoethyl)ethoxy)ethyl)]-5-methyluridine

To 1.3 mmol of2′-O-[2(2-(bis-N,N-dimethylaminoethyl-ethoxy)ethyl)]-5-methyl uridine inanhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytritylchloride (DMT-Cl, 0.87 g, 2 eq.) are added with stirring for 1 hour. Thereaction mixture is poured into water (200 mL) and extracted with CH₂Cl₂(2×200 mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

EXAMPLE 215′-O-Dimethoxytrityl-2′-O-[2(2-(bis-N,N-dimethylaminoethyl)ethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl) phosphoramidate

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-(bis-N,N-dimethylaminoethyl)ethoxy)ethyl)]-5-methyluridine(3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere of argon. Thereaction mixture is stirred overnight and the solvent evaporated. Theresulting residue is purified by silica gel flash column chromatographywith ethyl acetate as the eluent to give title compound.

EXAMPLE 225′-O-Dimethoxytrityl-2′-O-[2(2-bis(N,N-dimethylaminoethyl)ethoxy)ethyl)]-5-methyl-uridine-3′-O-succinate

5′-O-Dimethoxytrityl-2′-O-[2(2-bis-N,N-dimethylaminoethylethoxy)ethyl)]-5-methyluridine(0.41 mmol) is heated with 68 mg of succinic anhydride (0.6 mmol),4-N,N-dimethylaminoethyl pyridine (24 mg) and Et₃N (56 μL) indichloroethane (1 mL) at 50° C. for 10 minutes in a Pyrex tube in aheating block. After cooling, the reaction mixture is diluted withmethylene chloride (20 mL) and washed with a 10% aqueous solution ofcitric acid (3×20 mL) followed by water. The resulting solution is driedover anhydrous Na₂SO₄ to give the title compound.

EXAMPLE 235′-O-Dimethoxytrityl-2′-O-[2(2-bis(N,N-dimethylaminoethyl-ethoxy)ethyl)]-5-methyl-uridine-3′-O-succinylcontrolled pore glass (CPG)

5′-O-Dimethoxytrityl-2′-O-[2(2-bis-N,N-dimethylamino-ethylethoxy)ethyl)]-5-methyl-uridine-3′-O-succinate(0.15 mmol, 2 eq.) is dried under high vacuum overnight. To this driedmaterial is added CPG (650 mg, 1 eq.), anhydrous DMF (2 mL),N-methylmorpholine (33 μL, 4 eq.) and 2-1H-benzotriazole-1-yl-1,1,3,3-tetramethyluroniumtetrafluoro-borate (TBTU, 48 mg, 2 eq.) isadded to the reaction mixture with shaking for 12 hours. The CPG is thenfiltered and washed with DMF, CH₂Cl₂, CH₃CN and Et₂O. Finally, it isdried and capped with acetic anhydride/Et₃N. The loading of the CPG isdetermined via the dimethoxytrityl assay method.

EXAMPLE 24 General procedures for oligonucleotide synthesis

Oligomeric compounds are synthesized on a PerSeptive Biosystems Expedite8901 Nucleic Acid Synthesis System. Multiple 1-mmol syntheses areperformed for each oligonucleotide. The 3′-end nucleoside containingsolid support is loaded into the column. Trityl groups are removed withtrichloroacetic acid (975 mL over one minute) followed by anacetonitrile wash. The oligonucleotide is built using a modified diesteror thioate protocol.

Phosphodiester protocol All standard amidites (0.1 M) are coupled over a1.5 minute time frame, delivering 105 μL material. All novel amiditesare dissolved in dry acetonitrile (100 mg of amidite/1 mL acetonitrile)to give approximately 0.08-0.1 M solutions. The 2′-modified amidite isdouble coupled using 210 μL over a total of 5 minutes. Total couplingtime is approximately 5 minutes (210 mL of amidite delivered).1-H-tetrazole in acetonitrile is used as the activating agent. Excessamidite is washed away with acetonitrile. (1S)-(+)-(10-camphorsulfonyl)oxaziridine (CSO, 1.0 g CSO/8.72 mL dry acetonitrile) is used to oxidize(3 minute wait step) delivering approximately 375 μL of oxidizer.Standard amidites are delivered (210 μL) over a 3-minute period.

Phosphorothioate protocol

The 2′-modified amidite is double coupled using 210 μL over a total of 5minutes. The amount of oxidizer, 3H-1,2-benzodithiole-3-one-1,1-dioxide(Beaucage reagent, 3.4 g Beaucage reagent/200 mL acetonitrile), is 225μL (one =minute wait step). The unreacted nucleoside is capped with a50:50 mixture of tetrahydrofuran/acetic anhydride andtetrahydrofuran/pyridine/1-methyl imidazole. Trityl yields are followedby the trityl monitor during the duration of the synthesis. The finalDMT group is left intact. After the synthesis, the contents of thesynthesis cartridge (1 mmole) is transferred to a Pyrex vial and theoligonucleotide is cleaved from the controlled pore glass (CPG) using30% ammonium hydroxide (NH₄OH, 5 mL) for approximately 16 hours at 55°C.

Oligonucleotide Purification

After the deprotection step, the samples are filtered from CPG usingGelman 0.45 μm nylon acrodisc syringe filters. Excess NH₄H is evaporatedaway in a Savant AS160 automatic speed vac. The crude yield is measuredon a Hewlett Packard 8452A Diode Array Spectrophotometer at 260 nm.Crude samples are then analyzed by mass spectrometry (MS) on a HewlettPackard electrospray mass spectrometer. Trityl-on oligomeric compoundsare purified by reverse phase preparative high performance liquidchromatography (HPLC). HPLC conditions are as follows: Waters 600E with991 detector; Waters Delta Pak C4 column (7.8×300 mm); Solvent A: 50 mMtriethylammonium acetate (TEA-Ac), pH 7.0; Solvent B: 100% acetonitrile;2.5 mL/min flow rate; Gradient: 5% B for first five minutes with linearincrease in B to 60% during the next 55 minutes. Fractions containingthe desired product/s (retention time=41 minutes for DMT-ON-16314;retention time=42.5 minutes for DMT-ON-16315) are collected and thesolvent is dried off in the speed vac. Oligomeric compounds aredetritylated in 80% acetic acid for approximately 60 minutes andlyophilized again. Free trityl and excess salt are removed by passingdetritylated oligomeric compounds through Sephadex G-25 (size exclusionchromatography) and collecting appropriate samples through a Pharmaciafraction collector. The solvent is again evaporated away in a speed vac.Purified oligomeric compounds are then analyzed for purity by CGE, HPLC(flow rate: 1.5 mL/min; Waters Delta Pak C4 column, 3.9×300 mm), and MS.The final yield is determined by spectrophotometer at 260 nm.+

Procedures

Procedure 1

ICAM-1 Expression

Oligonucleotide Treatment of HUVECs: Cells were washed three times withOpti-MEM (Life Technologies, Inc.) prewarmed to 37° C. Oligomericcompounds were premixed with 10 μg/mL Lipofectin (Life Technologies,Inc.) in Opti-MEM, serially diluted to the desired concentrations, andapplied to washed cells. Basal and untreated (no oligonucleotide)control cells were also treated with Lipofectin. Cells were incubatedfor 4 h at 37° C., at which time the medium was removed and replacedwith standard growth medium with or without 5 mg/mL TNF-A α ® & DSystems). Incubation at 37° C. was continued until the indicated times.

Quantitation of ICAM-1 Protein Expression by Fluorescence-activated CellSorter: Cells were removed from plate surfaces by brief trypsinizationwith 0.25% trypsin in PBS. Trypsin activity was quenched with a solutionof 2% bovine serum albumin and 0.2% sodium azide in PBS (+Mg/Ca). Cellswere pelleted by centrifugation (1000 rpm, Beckman GPR centrifuge),resuspended in PBS, and stained with 3 μl/10⁵ cells of the ICAM-1specific antibody, CD54-PE (Pharmingin). Antibodies were incubated withthe cells for 30 min at 4° C. in the dark, under gently agitation. Cellswere washed by centrifugation procedures and then resuspended in 0.3 mLof FacsFlow buffer (Becton Dickinson) with 0.5% formaldehyde(Polysciences). Expression of cell surface ICAM-1 was then determined byflow cytometry using a Becton Dickinson FACScan. Percentage of thecontrol ICAM-1 expression was calculated as follows:[(oligonucleotide-treated ICAM-1 value)-(basal ICAM-1value)/(non-treated ICAM-1 value)-(basal ICAM-1 value)]. (Baker, Brenda,et. al. 2′-O-(2-Methoxy)ethyl-modified Anti-intercellular AdhesionMolecule 1 (ICAM-1) Oligomeric compounds Selectively Increase the ICAM-1mRNA Level and Inhibit Formation of the ICAM-1 Translation InitiationComplex in Human Umbilical Vein Endothelial Cells, The Journal ofBiological Chemistry, 272, 11994-12000, 1997.)

ICAM-1 expression of2′-O-[2-(2-N,N-dimethylaminoethyl)oxyethyl]-5-methyl modified oligomericcompounds of the invention is measured by the reduction of ICAM-1 levelsin treated HUVEC cells. The oligomeric compounds are believed to work bya direct binding RNase H independent mechanism. Appropriate scrambledcontrol oligomeric compounds are used as controls. They have the samebase composition as the test sequence.

Sequences that contain the2′-O-[2-(2-N,N-dimethylaminoethyl)oxyethyl]-5-methyl modification aslisted in Table 1 below are prepared and tested in the above assay. SEQID NO: 3, a C-raf targeted oligonucleotide, is used as a control.

TABLE 1 Oligomeric compounds Containing 2′-O-[2-(2-N,N- dimethylaminoethyl)oxyethyl]-5-methyl modification SEQ ID NO: Sequence (5′-3′)Target 1 5′-T _(S) C ^(m) _(s) T _(S) G _(s) A _(S) G _(s) T _(S) A _(s)G _(s) C ^(m) _(s) Human A _(s) G _(s) A _(s) G _(s) G _(S) A _(s) G_(S) C ^(m) _(s) T _(s) C-3′ ICAM-1 2 5′-T _(o) C ^(m) _(o) T _(o) G_(o) A _(o) G _(o) T _(o) A _(o) G _(o) C ^(m) _(o) Human A _(o) G _(o)A _(o) G _(o) G _(o) A _(o) G _(o) C ^(m) _(o) T _(o) C-3′ ICAM-1 3 5′-A_(s) T _(s) G _(s) C ^(m) _(s) A _(s) T _(s)T_(s)C_(s)^(m)T_(s)G_(s)C_(s) ^(m)C_(s) ^(m)C_(s) ^(m)C^(m) _(s) mouse C ^(m) _(s)A _(s) A _(s) G _(s) G _(s) A-3′ C-raf 4 5′-G_(s)C^(m) _(s)C^(m)_(s)C^(m) _(s)A_(s)A_(s)G_(s)C^(m) _(s)T_(s)G_(s)G_(s)C^(m) _(s) Human A_(S) T _(s) C ^(m) _(S) C ^(m) _(s) G _(S) T _(s) C ^(m) _(s) A-3′ICAM-1

All nucleosides in bold are2′-O-[2-(2-N,N-dimethylaminoethyl)oxyethyl]-5-methyl; subscript Sindicates a phosphorothioate linkage; subscript O indicates aphosphodiester linkage; and a superscript m on a C (C^(m)) indicates a5-methyl-C.

Procedure 2

Enzymatic Degradation of 2′-O-modified Oligomeric Compounds

Three oligomeric compounds are synthesized incorporating themodifications to the 3′ nucleoside at the 2′-O- position (Table 2).These modified oligomeric compounds are subjected to snake venomphosphodiesterase to determine their nuclease resistance. Oligomericcompounds (30 nanomoles) are dissolved in 20 mL of buffer containing 50mM Tris-HCl pH 8.5, 14 mM MgCl₂, and 72 mM NaCl. To this solution 0.1units of snake-venom phosphodiesterase (Pharmacia, Piscataway, N.J.), 23units of nuclease P1 (Gibco LBRL, Gaithersberg, Md.), and 24 units ofcalf intestinal phosphatase (Boehringer Mannheim, Indianapolis, Ind.)are added and the reaction mixture is incubated at 37° C. for 100 hours.HPLC analysis is carried out using a Waters model 715 automaticinjector, model 600E pump, model 991 detector, and an Alltech (AlltechAssociates, Inc., Deerfield, Ill.) nucleoside/nucleotide column (4.6×250mm). All analyses are performed at room temperature. The solvents usedare A: water and B: acetonitrile. Analysis of the nucleoside compositionis accomplished with the following gradient: 0-5 min., 2% B (isocratic);5-20 min., 2% B to 10% B (linear); 20-40 min., 10% B to 50% B. Theintegrated area per nanomole is determined using nucleoside standards.Relative nucleoside ratios are calculated by converting integrated areasto molar values and comparing all values to thymidine, which is set atits expected value for each oligomer.

TABLE 2 Relative Nuclease Resistance of 2′-Modified Chimeric Oligomericcompounds SEQ ID NO 5; 5′-TTT TTT TTT TTT TTT T*T*T*T*-3′ (Uniformphosphodiester) T* = 2′-modified T 2′-O-Modification —O—CH₂—CH₂—CH₃ Pr—O—CH₂—CH₂—O—CH₃ MOE —O-(DMAEOE) DMAEOE

Procedure 3

General Procedure for the Evaluation of Gapped 2′-O-DMAEOE ModifiedOligomeric Compounds Targeted to Ha-ras

Different types of human tumors, including sarcomas, neuroblastomas,leukemias and lymphomas, contain active oncogenes of the ras genefamily. Ha-ras is a family of small molecular weight GTPases whosefunction is to regulate cellular proliferation and differentiation bytransmitting signals resulting in constitutive activation of ras areassociated with a high percentage of diverse human cancers. Thus, rasrepresents an attractive target for anticancer therapeutic strategies.

SEQ ID NO: 6 is a 20-base phosphorothioate oligodeoxy-nucleotidetargeting the initiation of translation region of human Ha-ras and it isa potent isotype-specific inhibitor of Ha-ras in cell culture based onscreening assays (IC₅₀=45 nm). Treatment of cells in vitro with SEQ IDNO: 6 results in a rapid reduction of Ha-ras mRNA and protein synthesisand inhibition of proliferation of cells containing an activating Ha-rasmutation. When administered at doses of 25 mg/kg or lower by dailyintraperitoneal injection (IP), SEQ ID NO: 6 exhibits potent antitumoractivity in a variety of tumor xenograft models, whereas mismatchcontrols do not display antitumor activity. SEQ ID NO: 6 has been shownto be active against a variety of tumor types, including lung, breast,bladder, and pancreas in mouse xenograft studies (Cowsert, L. M.Anti-cancer drug design, 1997, 12, 359-371). A second-generation analogof SEQ ID NO: 6, where the 5′ and 3′ termini (“wings”) of the sequenceare modified with 2′-methoxyethyl (MOE) modification and the backbone iskept as phosphorothioate (Table 2, SEQ ID NO: 12), exhibits IC₅₀ of 15nm in cell culture assays. Thus, a 3-fold improvement in efficacy isobserved from this chimeric analog. Because of the improved nucleaseresistance of the 2′-MOE phosphorothioate, SEQ ID NO: 12 increases theduration of antisense effect in vitro. This will relate to frequency ofadministration of this drug to cancer patients. SEQ ID NO: 12 iscurrently under evaluation in ras dependent tumor models (Cowsert, L. M.Anti-cancer drug design, 1997, 12, 359-371). The parent compound, SEQ IDNO: 6, is in Phase I clinical trials against solid tumors by systemicinfusion. Antisense oligomeric compounds having the 2′-O-DMAEOEmodification are prepared and tested in the aforementioned assays in themanner described to determine activity. Oligomeric compounds that areinitially prepared are listed in Table 3 below.

TABLE 3 Ha-ras Antisense Oligomeric compounds With 2′-O-DMAEOEModifications and Their Controls SEQ ID Back- 2′- Com- NO: Sequence boneModif. ments 6 5′-TsCsCs GsTsCs AsTsCs Gs P=S 2′-H parent CsTs CsCsTsCsAsGs GsG-3′ 7 5′-TsCsAs GsTsAs AsTsAs Gs P=S 2′-H mismatch GsCs CsCsAsCsAsTs GsG-3′ control 8 5′-ToToCo GsTsCs AsTsCs Gs  P=O/ 2′-O- GapmerCsTs CoCoTo CoAoGo GoG-3′ P=S/ DMAEOE (mixed P=S in the back- wingsbone) 9 5′-TsCsCs GsTsCs AsTsCs Gs  P=S 2′-O- Gapmer CsTs CsCsTs CsAsGsGsG-3′ DMAEOE as in the uniform wings thioate 10 5′-ToCoAoGsTsAs AsTsAs  P=O/ 2′-O- Gapmer GsCsCs GsCsCs Gs Co P=S/ DMAEOE (mixedCo CoCoAo CoAoTo GoG-3′ P=O in the back- wings bone) 11 5′-TsCsAsGsTsAs AsTs  P=S 2′-O- Gapmer As GsCsCs GsCsCs DMAEOE as CsCsAs CsAsTsGsC-3′ in the uniform wings thioate 12 5′-TsCsCs GsTsCs AsTsCs Gs  P=S2′-MOE Gapmer CsTs CsCsTs CsAsGs GsG-3′ in the with wings MOE as control13 5′-TsCsAsGsTsAs AsTsAsGsCs  P=S 2′-MOE Gapmer CsGsCsCsCsCsAsCsAsTsGsC-3′ in the with wings MOE as control underlined portions of sequencesare 2′-deoxy

Procedure 4

General Procedure for the Evaluation of 2′-O-DMAEOE Oligomeric CompoundsTargeted to HCV

Uniformly modified 2′-O-DMAEOE phosphodiester oligomeric compounds areevaluated for antisense inhibition of HCV gene via a translation arrestmechanism.

Hepatitis C virus (HCV) is known to be responsible for liver disease inmany millions of people throughout the world. HCV is an enveloped,positive-strand RNA virus of the flavivirus family. Initial infectionsin humans are typically asymptomatic, but chronic infection often ensuesin which liver cirrhosis and hepatocellular carcinoma are long-termsequelae. Interferon-α (IFN-α) therapy is widely used in attempts toeradicate the virus from chronically infected individuals, but long-termremissions are achieved in only about 20% of patients, even after 6months of therapy. So far, there is no antiviral drug available for thetreatment of HCV. (Blair et al., 1998). Drug discovery and developmentefforts have been hampered by the lack of suitable cell culturereplication assays for HCV, and vaccine production has been hampered bygenetic variability of the virus' envelope genes. Specific inhibitors ofcloned viral enzymes such as proteases and the viral polymerase have notyet been reported.

Antisense oligonucleotide therapy represents a novel approach to thecontrol of HCV infection. Several antisense oligomeric compoundscomplementary to HCV RNA sequences adjacent to the polyproteininitiation codon of HCV have been designed at Isis (Hanecak et al., J.Virol., 1996, 70, 5203-5212). The target genome is highly conservedamong independent HCV isolates.

It was shown that an RNase H-independent antisense oligonucleotide hadgreater activity than its parent phosphorothioate (which will work byRNase H mechanism) which was targeted to the AUG site of a core proteinsequence of HCV in a human hepatocyte cell line employing a uniformlymodified 2′-O-(methoxyethyl) phosphodiester (P=O 20 mer) (Hanecak etal., J. Virol., 1996, 70, 5203-5212). Hepatitis C virus core proteinlevels were reduced as efficiently as the corresponding2′-deoxyphosphorothioate with an IC₅₀ of 100 nm. SEQ ID NO: 15 was apotent inhibitor of core protein expression without affecting HCV RNAlevels. This suggested the inhibition of HCV translation. The parentcompound (SEQ ID NO: 14) had Tm of 50.8° C. while the 2′-MOE compound(SEQ ID NO: 15) had a Tm of 83.8° C. Thus, SEQ ID NO: 15 had a betteraffinity for HCV RNA. The replicative cycle of HCV takes place in thecytoplasm of infected cells, in which RNase H levels have been reportedto reduce relative to those of the nucleus. For this reason, it isbetter to utilize an antisense oligonucleotide which will work bynon-RNase H mechanism to inhibit HCV. Oligonucleotide SEQ ID NO: 15 isan attractive lead since it contains a P=O linkage with a 21′-MOEmodification. SEQ ID NO: 16 will be tested in accordance with thetesting of SEQ ID NO: 14 and 15.

TABLE 4 5′-TTT AGG ATT CGT GCT CAT GG-3′ Antisense OligonucleotideTargeting HCVC 5′-NCR Nucleotide Numbers 340-359 SEQ ID NO: Backbone2′-modification Tm (° C.) 14 P=S 2′-deoxy 50.8 15 P=O 2′-MOE 83.8 16 P=O2′-2′-O-DMAEOE

Procedure 5

In vitro Assays

Isis antisense oligomeric compounds complementary to the HCV polyproteininitiation codon sequence are known to inhibit expression of the viralcore protein in immortalized cell lines engineered to express HCV RNAfrom recombinant DNA integrated into the host cell genome (Hanecakibid). Non-complementary control oligomeric compounds have no effect onHCV RNA or protein levels in this system. H8Ad17C cells will be treatedwith a range of concentration of oligomeric compounds shown in Table 4above, especially SEQ ID NO: 16, (0-200 nm) in the presence of cationiclipids and total protein levels will be evaluated 20 hours later bywestern blot analysis.

Procedure 6

In vivo Model for HCV

Animal models of HCV infection are not readily available. An alternativeapproach has been developed to evaluate antisense oligomeric compoundsto inhibit HCV gene expression in livers of mice. For these experiments,HCV sequences, including SEQ ID NO: 15 target sequence, were fused to aluciferase reporter gene and inserted into a Vaccinia virus. Infectionof mice with this recombinant vaccination virus results in quantifiablelevels of luciferase in liver tissue. Potent phosphorothioate antisenseoligomeric compounds have been shown to work in this model. SEQ ID NO:16 (the 2′-O-DMAEOE RNA analog of SEQ ID NO: 15) will be evaluated forinhibition of expression of the HCV-luciferase construct in livers ofmice infected with the recombinant vaccinia virus. Inhibition will beevaluated for sequence-dependency and dose response. HCV-luciferaseexpression in livers of mice infected with a control vaccinia virusvector lacking HCV target sequences will be used as control and theeffect of antisense drug in these control systems will be evaluated.(Antisense oligonucleotide-mediated inhibition of hepatitis C virus geneexpression in mouse liver (Anderson et al., Meeting Abstracts,International Hepatitis Meeting, Hawaii, 1997).

Procedure 7

In vivo Nuclease Resistance

The in vivo Nuclease Resistance of gapmers having the 2′-O-DMAEOE isstudied in mouse plasma and tissues (kidney and liver). For thispurpose, the C-raf oligonucleotide series SEQ ID NO: 17 will be used andthe following five oligomeric compounds listed in Table 5 below will beevaluated for their relative nuclease resistance.

TABLE 5 SEQ ID NO: Sequence Backbone Description 17 5′-ATG CAT TCT GCCP=S, (control) CCA AGGA-3′ 2′-H rodent C-raf antisense oligo 18AoToGoCoAsTsTsCsTsGs P=O/P=S/ (control) CsCsCsCsAoAoGoGoA  P=O2′-MOE/2′-H/ 2′-MOE  19 AsTsGsCsAsTsTsCsTsGs P=S (control)CsCsCsCsAsAsGsGsA  2′-MOE/2′-H/2′- MOE 20 AoToGoCoA sTsTsCsTsGs P=O/P=S/2′-2′-O-DMAEOE / CsCsCsCs AoAoGoGoA   P=O 2′-H/ 2′-2′-O-   DMAEOE   21AsTsGsCsA sTsTsCsTsGs P=S 2′-2′-O-DMAEOE / CsCsCsCs AsAsGsGsA   2′-H/2′-2′-O-   DMAEOE

Procedure 8

Animal Studies

For each oligonucleotide to be studied, 9 male BALB/c mice (CharlesRiver, Wilmington, Mass.), weighing about 25 g are used (Crooke et al.,J. Pharmacol. Exp. Ther., 1996, 277, 923). Following a 1-weekacclimation, the mice receive a single tail vein injection ofoligonucleotide (5 mg/kg) administered in phosphate buffered saline(PBS), pH 7.0. The final concentration of oligonucleotide in the dosingsolution is (5 mg/kg) for the PBS formulations. One retro-orbital bleed(either 0.25, 9.05, 2 or 4 post dose) and a terminal bleed (either 1, 3,8 or 24 h post dose) is collected from each group. The terminal bleed(approximately 0.6-0.8 mL) is collected by cardiac puncture followingketamine/xylazine anesthesia. The blood is transferred to an EDTA-coatedcollection tube and centrifuged to obtain plasma. At termination, theliver and kidneys will be collected from each mouse. Plasma and tissueshomogenates will be used for analysis for determination of intactoligonucleotide content by CGE. All samples will be immediately frozenon dry ice after collection and stored at −80° C. until analysis.

Procedure 9

The binding affinity as measured by Tm was evaluated for oligomericcompounds having the 2′-O-[2-(2-N,N-dimethylaminoethyl)oxyethyl]modification. 5-methyl-2′-O-[2-(2-N,N-dimethylaminoethyl)oxyethyl](modified T) was incorporated at selected positions in oligonucloetidesand binding was measured to complementary RNA oligomeric compounds.

TABLE 6 SEQ ID Mass NO: Sequence Backbone Calculated Observed 22 TCC AGGTGT CCG PO 5660.3^(a) 5659.l^(a) CAT C 23 CTC GTA CTT TTC PO 5912.25913.5 CGG TCC 24 GCG TTT TTT TTT  PO 6487.3^(a) 6488.5^(a) TGC G 25GAT CT PO 1910.6^(a) 1910.8^(a) 26 TTT TTT TTT TTT PO 6544.7^(a)6542.62^(a) TTT TTT T  ^(a)as DMT-on, underlined nucleosides are2′-O-[2-(2-N,N-dimethylaminoethyl)oxyethyl]-5-methyl uridine (2′ -sub-T)

TABLE 7 Tm values SEQ Tar- Tar- ID get ΔTm/ get ΔTm/ NO: Sequence DNAΔTm mod. RNA ΔTm mod. 27 TCC AGG TGT CCG not determined 62.3 CAT C 22TCC AGG TGT CCG not determined 65.6 3.3 0.83° CAT C 28 GCG TTT TTT TTT54.2 48.1 TGC G 24 GCG TTT TTT TTT  50.0 −4.1 −4.1 59.7 10.6 1.1° TGC G

Underlined nucleosides are 2′-O-[2-(2-N,N-dimethylaminoethyl)oxyethyl]modified.

The 2′-O-[2-(2-N,N-dimethylaminoethyl)oxyethyl] modified nucleosidicmonomers show increased Tm as compared to unmodified DNA as shown inTable 7.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

28 1 20 DNA Artificial Sequence Synthetic construct 1 tctgagtagcagaggagctc 20 2 20 DNA Artificial Sequence Synthetic construct 2tctgagtagc agaggagctc 20 3 20 DNA Artificial Sequence Syntheticconstruct 3 atgcattctg cccccaagga 20 4 20 DNA Artificial SequenceSynthetic construct 4 gcccaagctg gcatccgtca 20 5 19 DNA ArtificialSequence Synthetic construct 5 tttttttttt ttttttttt 19 6 20 DNAArtificial Sequence Synthetic construct 6 tccgtcatcg ctcctcaggg 20 7 20DNA Artificial Sequence Synthetic construct 7 tcagtaatag gcccacatgg 20 820 DNA Artificial Sequence Synthetic construct 8 ttcgtcatcg ctcctcaggg20 9 20 DNA Artificial Sequence Synthetic construct 9 tccgtcatcgctcctcaggg 20 10 26 DNA Artificial Sequence Synthetic construct 10tcagtaatag ccgccgcccc acatgg 26 11 23 DNA Artificial Sequence Syntheticconstruct 11 tcagtaatag ccgccccaca tgc 23 12 20 DNA Artificial SequenceSynthetic construct 12 tccgtcatcg ctcctcaggg 20 13 23 DNA ArtificialSequence Synthetic construct 13 tcagtaatag ccgccccaca tgc 23 14 20 DNAArtificial Sequence Synthetic construct 14 tttaggattc gtgctcatgg 20 1520 DNA Artificial Sequence Synthetic construct 15 tttaggattc gtgctcatgg20 16 20 DNA Artificial Sequence Synthetic construct 16 tttaggattcgtgctcatgg 20 17 19 DNA Artificial Sequence Synthetic construct 17atgcattctg ccccaagga 19 18 19 DNA Artificial Sequence Syntheticconstruct 18 atgcattctg ccccaagga 19 19 19 DNA Artificial SequenceSynthetic construct 19 atgcattctg ccccaagga 19 20 19 DNA ArtificialSequence Synthetic construct 20 atgcattctg ccccaagga 19 21 19 DNAArtificial Sequence Synthetic construct 21 atgcattctg ccccaagga 19 22 16DNA Artificial Sequence Synthetic construct 22 tccaggtgtc cgcatc 16 2318 DNA Artificial Sequence Synthetic construct 23 ctcgtacttt tccggtcc 1824 16 DNA Artificial Sequence Synthetic construct 24 gcgttttttt tttgcg16 25 5 DNA Artificial Sequence Synthetic construct 25 gatct 5 26 19 DNAArtificial Sequence Synthetic construct 26 tttttttttt ttttttttt 19 27 16DNA Artificial Sequence Synthetic construct 27 tccaggtgtc cgcatc 16 2619 DNA Artificial Sequence Synthetic construct 28 gcgttttttt tttgcg 16

What is claimed is:
 1. An oligomeric compound having at least onenucleoside of the formula:

wherein Bx is a heterocyclic base; each R₁ and R₂ is, independently, H,a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted C₂-C₁₀ alkenyl, substituted orunsubstituted C₂-C₁₀ alkynyl, wherein said substitution is OR₃, SR₃, NH₃⁺, N(R₃)(R₄), guanidino or acyl where said acyl is an acid, amide or anester; or R₁ and R₂, together, are a nitrogen protecting group or arejoined in a ring structure that optionally includes an additionalheteroatom selected from N and O; and each R₃ and R₄ is, independently,H, C₁-C₁₀ alkyl, a nitrogen protecting group, or R₃ and R₄, together,are a nitrogen protecting group; or R₃ and R₄ are joined in a ringstructure that optionally includes an additional heteroatom selectedfrom N and O.
 2. The oligomeric compound of claim 1 wherein R₁ is H,C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl and R₂ is C₁-C₁₀ substitutedalkyl.
 3. The oligomeric compound of claim 2 wherein R₁ is C₁-C₁₀ alkyl.4. The oligomeric compound of claim 2 wherein R₂ is C₁-C₁₀ substitutedalkyl and the substituent is NH₃ ⁺ or N(R₃) (R₄).
 5. The oligomericcompound of claim 2 wherein R₁ and R₂ are both C₁-C₁₀ substituted alkyl.6. The oligomeric compound of claim 5 wherein said substituents are,independently, NH₃ ⁺ or N(R₃) (R₄).
 7. The oligomeric compound of claim1 wherein R₁ and R₂ are each C₁-C₁₀ alkyl.
 8. The oligomeric compound ofclaim 1 wherein R₁ and R₂ are joined in a ring structure that caninclude at least one heteroatom selected from N and O.
 9. The oligomericcompound of claim 8 wherein said ring structure is imidazole,piperidine, morpholine or a substituted piperazine.
 10. The oligomericcompound of claim 9 wherein the substituent on said piperazine is C₁-C₁₂alkyl.
 11. The oligomeric compound of claim 1 wherein said heterocyclicbase is a purine or a pyrimidine.
 12. The oligomeric compound of claim11 wherein said heterocyclic base is adenine, cytosine,5-methylcytosine, thymine, uracil, guanine or 2-aminoadenine.
 13. Theoligomeric compound of claim 1 comprising from about 5 to about 50nucleosides.
 14. The oligomeric compound of claim 1 comprising from bout8 to about 30 nucleosides.
 15. The oligomeric compound of claim 1comprising from about 15 to about 25 nucleosides.
 16. A compound of theformula:

wherein: Bx is a heterocyclic base; T₁ and T₂, independently, are OH, aprotected hydroxyl, an activated phosphorus group, a reactive group forforming an internucleotide linkage, a nucleoside, a nucleotide, anoligonucleoside an oligonucleotide or a linkage to a solid support; eachR₁ and R₂ is, independently, H, a nitrogen protecting group, substitutedor unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein saidsubstitution is OR₃, SR₃, NH₃ ⁺, N(R₃) (R₄), guanidino or acyl wheresaid acyl is an acid, amide or an ester; or R₁ and R₂, together, are anitrogen protecting group or are joined in a ring structure thatoptionally includes an additional heteroatom selected from N and O; andeach R₃ and R₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protectinggroup, or R₃ and R₄, together, are a nitrogen protecting group; or R₃and R₄ are joined in a ring structure that optionally includes anadditional heteroatom selected from N and O.
 17. The compound of claim16 wherein R₁ is H, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl and R₂ isC₁-C₁₀ substituted alkyl.
 18. The compound of claim 17 wherein R₁ isC₁-C₁₀ alkyl.
 19. The compound of claim 17 wherein R₂ is C₁-C₁₀substituted alkyl and the substituent is NH₃ ⁺ or N(R₃) (R₄).
 20. Thecompound of claim 17 wherein R₁ and R₂ are both C₁-C₁₀ substitutedalkyl.
 21. The compound of claim 20 wherein said substituents are,independently, NH₃ ⁺ or N(R₃) (R₄).
 22. The compound of claim 16 whereinR₁ and R₂ are each C₁-C₁₀ alkyl.
 23. The compound of claim 16 wherein R₁and R₂ are joined in a ring structure that can include at least oneheteroatom selected from N and O.
 24. The compound of claim 23 whereinsaid ring structure is imidazole, piperidine, morpholine or asubstituted piperazine.
 25. The compound of claim 24 wherein saidsubstituted piperazine is substituted with a C₁-C₁₂ alkyl.
 26. Thecompound of claim 25 wherein said heterocyclic base is a purine or apyrimidine.
 27. The compound of claim 26 wherein said heterocyclic baseis adenine, cytosine, 5-methylcytosine, thymine, uracil, guanine or2-aminoadenine.
 28. The compound of claim 16 wherein T₁ is a hydroxylprotecting group.
 29. The compound of claim 16 wherein T₂ is anactivated phosphorus group or a connection to a solid support.
 30. Thecompound of claim 29 wherein said solid support material ismicroparticles.
 31. The compound of claim 29 wherein said solid supportmaterial is CPG.